This invention relates to methods for treating various diseases, specifically cancer, using a targeted drug delivery nanocarrier that is selected to specifically and selectively bind to a target site. More particularly, this invention relates engineered tumor-targeted drug nanocarriers with controlled specificity, stability and high loading efficiency, suitable for the targeted intra-tumoral and intracellular delivery of pharmaceuticals.
Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. If the spread is not controlled, it can result in death. Cancer is caused by both external factors (e.g. tobacco, chemicals, radiation and infectious organisms) and internal factors (inherited mutations, immune system conditions, the mutations that occur from metabolism). These causal factors may act together or in sequence to initiate or promote carcinogenesis. Currently, cancer is treated by surgery, radiation, chemotherapy, hormones and immunotherapy. However, there is an urgent need for more effective anti-tumor cancer drugs. For example, the life time risk for clinical prostate cancer is about 10% among U.S. men; approximately 3% die of this disease. Despite advances in early detection and treatment of the disease, the mortality rate has not declined, indicating that the current therapies are not adequate and new strategies are required.
The ideal anti-tumor therapy would enable the delivery of highly cytotoxic agent specifically to tumor cells and would leave normal cells unaffected. Conventional chemotherapeutic treatment, for example, with the agent doxorubicin, is limited because of the toxic side-effects that arise. The idea of drug targeting was first suggested by Paul Ehrlich more than 100 years ago. Recently, several approaches have been provided for the creation of tumor-targeted drugs.
One approach utilized conjugates of tumor-cystic probes with toxins, McCune et al., Journal of the American Medical Association 286, 1149-1152 (2001); Wahl, et al. Int. J. Cancer 1993, 590-600 (2001). For example, monoclonal antibodies or growth factors, such as epidermal growth factor (EGF) were conjugated to various toxins including pseudomonas or diphtheria toxins, which arrest the synthesis of proteins and cells, see, e.g., FitzGerald and Pastan, Journal of the National Cancer Institute 81, 1455-1463 (1989). However, the disadvantage of this type of system is that it may provoke an immune system reaction due to the non-human components, which decreases the effectiveness of the treatment and may result in a suppression of the immune system. Additionally, the drug conjugates are subject to elimination from the circulation through renal filtration, and schematic degradation, uptake by the reticuloendothelial system (RES) and accumulation in non-targeted organs and tissues.
Another approach takes the advantage of the hyper-permeability of vascular endothelia at tumor sites by using passive drug carriers, such as polymers, see e.g. Kostarelos and Emfietzoglou, Anti Cancer Research 20, 3339-3345 (2000); Matsumura and Maeda, Cancer research 46, 6387-6392, (1986); and thanou and duncan, Curr. Opin. Investig. Drugs 4, 701-709 (2003). Other passive drug carriers suggested by the literature included liposomes and polymeric micelles, Duncan et al., J. Control release 74, 135-146 (2001); Husseini et al., J. Control release 83, 303-305 (2002); Kataoka et al., J. Control release 64, 143-153 (2000); Rapoport et al., J. Control release 91, 85-95 (2003). Matsumura and Maeda, cited above, observed that polymeric drugs and macromolecules accumulate within solid tumors due to an enhanced permeability and retention mechanism. The enhanced permeability and retention mechanism is based on characteristics of solid tumors such as high vascular density, reduced lymphatic drainage, extensive production of vascular mediators and defects in vascular structure.
Accordingly, “magic shells” of individual drug molecules packed into targeted carriers that protect the drug molecules from inactivation in an aggressive biological environment and improve drug delivery to the site of disease are considered the state of the art in drug delivery systems. In order to perform its mission and affect cancer cells in a tumor, a blood-borne therapeutic particle must travel into the blood vessels of the tumor, pass across the vessel wall into the interstitium, migrate through the interstitium, and unload its cargo into the tumor cells. Organ or tissue accumulation may be achieved by the passive targeting via the enhanced permeability and retention of the tumoral tissue or by active probe-mediated targeting. Intracellular delivery may be mediated by cell-recognizing and penetrating ligands.
The concept of targeted drug nanocarriers has stimulated tremendous research efforts and resulted in designs of new carrier particles, such as micelles, liposomes, capsules, spheres, etc. and their conversion into physiologically acceptable and stable drug carriers, Torchilin, Nat. Rev. Drug discov. 4, 145-160 (2005); Churchland et al., Proc. Nat'l. Acad. Sci. USA 100, 6039-6044 (2003). Micelles and liposomes will be further discussed, herein. Despite the recent advances, there are still some physiological barriers in realizing the concept of targeted drug carriers. These barriers include fast clearance of foreign particles from the blood, and technological hindrances in obtaining highly standardized, pharmaceutically acceptable multi-functional nanoparticles. The biggest challenge, however, is that particles are still mostly administered through circulation. In order to stimulate accumulation of the drug loaded nanocarriers at the target site, the nanocarriers should be supplied with specific probes capable of binding the target tumor cells. Such nanocarriers need to have longevity and target recognition. Attempts have been made to conjugate micelles and liposomes with water soluble polymers and target specific probes. However, the majority of these particles are still cleared through circulation because the probes lack the specificity and selectivity necessary for high efficacy in administration of the drug to the target site.
The size and surface properties of the carrier particles are of crucial importance in achieving controlled drug delivery. Ideally, carrier particles should be small biodegradable particles with good loading capacity, prolonged circulation, and ability to accumulate in required areas. These requirements are reasonably well-met by micelles and liposomes, which are well-known in the art for use in poorly soluble and water-soluble drugs.
Micelles are self-assembling spherical colloidal nanoparticles formed by amphiphilic molecules. Micelles are also described as aggregate surfactant molecules disbursed in a liquid colloid. As demonstrated in FIG. 1, hydrophobic fragments 1 of amphiphilic molecules form the core of a micelle while their hydrophilic heads 3 form a micelle corona. The core of the micelle, which is segregated in an aqueous milieu, is capable of encapsulating drugs protecting them from destruction and biological surroundings while improving their pharmacokinetics and biodistribution. Micelles are generally in the order of 5-50 nm in diameter, and are therefore capable of accumulating in pathological areas with leaky vasculature, such as infarct zones and tumors due to the enhanced permeability and retention effect. Micelles are also capable of evading a major obstacle in drug targeting by particulate systems: non-specific uptake by the reticulo-endothelial systems and renal secretion.
Micelles may be formed by any of commonly known surfactants, such as sodium dodecylsulfate or phospholipids, but the performance of such surfactants as drug delivery systems is low compared to micelles composed of specially designed block copolymers, as described in Kataoka et al., supra and Torchilin et al., supra (2003). The flexible hydrophilic polymers, which are used as shell-forming segments for the polymer micelles, assemble into a dense palisade shell, which is cross-linked by numerous water molecules to achieve effective stabilization of the vesicle. Accordingly, the polymer micelles dissociate much more slowly than unmodified surfactant micelles, retain the loaded drugs for a longer period of time and accumulate the drug at the target site more efficiently. Further, polymer micelles are readily engineered to have sizes in the range of several tens of nanometers with a narrow size distribution which is a great advantage in regulating biodistribution.
In contrast to micelles, liposomes are a bilayered phospholipid vesicles approximately 50 to 1,000 nm in diameter. As shown in FIG. 2, liposomes can carry a variety of water soluble and water insoluble drugs loaded in an inner aqueous compartment 2 or into the phospholipid bilayer 4. Liposomes are biologically inert and completely biocompatible; they cause practically no toxic or antigenic reactions. Drugs included into liposomes are protected from the destructive action of the external media by the liposomes. Thus, liposomes are able to deliver their content inside cells and even inside different cell compartments. Water-soluble drugs can be captured by the inner aqueous compartment of liposomes, whereas lipophilic compounds can be incorporated into the phospholipid bilayer. Like drug loaded micelles, drug loaded liposomes rely on passive targeting and the enhanced permeability and retention effect that allows for the accumulation of anti-cancer drugs in the solid tumors without affecting normal tissues. The differential accumulation of micelle and liposomal drugs in tumor tissues relative to normal tissues is the basis for increased tumor specificity relative to free drugs. Accordingly, liposomes are considered a promising drug carrier with significant therapeutic potential, as demonstrated in numerous laboratory tests and clinical trials, e.g., Torchilin, Nat. Rev. Drug discov. 4, 145-160 (2005).
It is known that liposomes and micelles can be stabilized by enhancing the outermost hydrophobic shell with water soluble polymers, such as polyethyleneglycol (PEG). The presence of hydrophilic polymers on the hydrophobic surface of these carrier particles attracts a water shell, resulting in reduced adsorption of opsonins to the carrier particles. This, in turn, results in a decrease in both the rate and extent of uptake of carrier particles by mononuclear phagocytes. Long circulating liposomes improved the therapeutic index of drugs and encapsulated therein. Currently, several preparations based on long circulating liposomes are commercially available, for example, Doxil®, a doxorubicin containing polyethyleneglycolated (PEGylated) liposomes, Sharp et al., Drugs 62 2089-2126 (2002). Doxil is manufactured by ortho biotech products, LP of bridgewater, N.J., USA. O'Shaughnessy, Clin. Breast cancer 4, 318-328, (2003), demonstrated selective delivery of doxorubicin into solid tumors in patients with breast carcinoma metastases was achieved by capsulation of the drug into PEGylated liposomes, which resulted in subsequent improvement of survival. Efficacy was also demonstrated by combining liposomal doxorubicin with paclitaxel (available as Taxol®, Bristol-Meyers Squibb Company, New York, N.Y., USA) caelyx (Schering-Plough corporation, Kenilworth, N.J., USA) and carboplatin (available as Paraplatin® from Bristol-Meyers Squibb company). Several preparations of liposomes have been approved for clinical application or undergoing clinical evaluation, Torchilin, supra, (2005).
It is also known in the art to encapsulate antibiotic and antibacterial drugs within carrier particles such as micelles or liposomes. Moreover, it is known in the art to include therapeudically active polynucleotides, e.g., RNA, DNA, cDNA, mRNA, etc., into liposomes for protected administration.
One of the distinct drawbacks of liposome and micelle preparations injected intravenously for systemic application is their fast elimination from the blood because of their capture by the cells of the reticulo-endothelial system, primary the liver. As aforementioned, this problem was first addressed by adhering water soluble polymers to the carrier particles' outer shell. Another solution is to target the effected organ or tissue by coupling the loaded carrier particle with ligands capable of recognizing and binding to cells of interest.
In order to achieve more specific targeting of carrier particles, such particles are modified with various ligands using advance conjugation procedures. For example, antibodies and small peptides have been attached to the water exposed tips of polyethyleneglycol chains, Blume, et al. Biomembranes 1149, 180-184 (1993). Antibodies and small peptides have also been conjugated via reactive p-nitrophenylcarbonyl, N-benzotrazole carbonyl or maleimide terminated PEG-phosphatidylethanolamine, Moreira, Pharm. Res. 19, 265-269 (2002); Torchilin et al., supra (2001); xiong, et al., J. Pharm. Sci. 94, 1782-1793 (2005). These conjugation procedures, which are adapted from the arsenal of organic chemistry, are effective for the preparation of various targeted carrier particles on a small scale basis, i.e., for preliminary laboratory and clinical studies, it would be significantly less efficient when moved to large scale preparation where standardized pharmaceutically acceptable preparations will be required. For example, it was noted in the most advanced recent studies, Nellis, et al., Biotechnol. Prog. 21, 205-220 (2005), that the largest 40-L culture produced enough of F5cys to manufacture 2,085 mg of conjugate, enough to support planned pre-clinical and future clinical trials. This extremely laborious procedure, including high volume propagation of bacteria, several chromatographic steps for producing the targeted ligand, sophisticated conjugation procedure and further chromatographic purification of the conjugated lipid moiety, yields a conjugate with only 93% purity. Obviously, this would be inefficient and highly cost expensive at the production stage.
Thus, despite its promise, targeted carrier particle technology is not without difficulties. Preparation of the targeting ligands, such as antibodies, and their conjugation to the lipids to make usable quantities of the targets of carriers has proven troublesome, differing idiosyncratically from one targeted particle to another. Accordingly, there is a need for an easily assembled targeted carrier particle that has efficient assembly/conjugation, little bioreactivity and specificity and selectivity in binding target sites.
To respond to the challenge of drug targeting, targeting technologies are being revolutionized by utilizing methods of combinatorial chemistry and phage display. The present inventor and colleagues have developed a phage display library where targeted peptides or antibodies are selected from billion clone phage display libraries and then expressed in bacteria or chemically synthesized to obtain a desired bioselective material, Petrenko and sorokulova, Journal of microbiological methods 58, 147-168 (2004); smith and petrenko, Chemical reviews 97, 391-410 (1997).
Phage-display libraries refer to a selection technique wherein a library of variants of a peptide or protein is expressed on the outside of a phage virion, while the genetic material encoding the peptide or protein remains inside the phage. Phage-display libraries are constructed by the genetic modification of filamentous bacterial viruses (phages) such as M13, f1, and fd. Referring now to FIG. 3, these bacteriophages are lengthy, their virions consisting of single stranded circular DNA packaged in a cylindrical shell of a major coat protein pVIII. The outer coats of these filamentous phages are composed of thousands of α-helical subunits of major coat protein pVIII which form a tube encasing the viral DNA. At the tips of the phage are several copies of each of the minor proteins, pIII, pVI, pVII, and pIX. To create a phage-display library, degenerate synthetic oligonucleotides are spliced in-frame into one of the phage coat protein genes, so that the peptide encoded by the degenerate oligonucleotide is fused to the coat protein and thereby displayed on the exposed surface of the phage virion. Accordingly, each phage virion displays multiple copies of one particular peptide.
Referring now to FIG. 4, in landscape phages, as in traditional phage-display constructs, foreign peptides or proteins 5 are fused to coat proteins 7 on the surface of the virus particle. Unlike conventional phage constructs, however, landscape phages display thousands of copies of the peptide 5 in a repeating pattern, comprising a major fraction of the viral surface. The phage body serves as an interacting scaffold to constrain the peptide into a particular confirmation, creating a defined organic surface structure, i.e., the landscape. The particular conformation, and thus organic surface structure, varies from one phage clone to the next accordingly, a landscape phage library is a huge population of such phages, encompassing billions of clones with different surface structures and biophysical properties.
The major coat protein pVIII is a typical membrane protein. During infection of a host, e.g., E. coli, with the filimentous bacteriophage, the coat is dissolved in the bacterial cytoplasmic membrane, while viral DNA enters the cytoplasm. Protein is synthesized in the infected cell as a water soluble cytoplasmic precursor, which contains an additional leader sequence of 23 residues at its N-terminus. When this protein is inserted into the membrane, the leader sequence is cleved off by a leader peptidase. Later, during the page assembly the newly synthesized major pVIII proteins are transferred from the membrane into the coat of the emerging phage. The structural flexibility of major coat protein is determined by its unique architecture. Thus, the major coat protein pVIII can change its confirmation to accommodate various distinct forms of the phage and its precursors: phage filament, intermediate particle form (I-form), spheroid form (S-form), and membrane bound form.
The ability of the major protein pVIII to become associated with micelles and liposomes emerges from its intrinsic function as a membrane protein. The structure of major coat protein pVIII in micelles and bilayer membranes is well resolved. A 50 amino acid long pVIII protein is very hydrophobic and insoluble in water when separated from virus particles or membranes. In virus particles, it forms a single, distorted α-helix with only the first four to five residues mobile and unstructured. It is arranged in layers with a five-fold rotational symmetry and approximately two-fold screw symmetry around the filament access, as demonstrated in FIG. 5.
Still referring to FIG. 5, in the membrane bound form of the pVIII protein, the 16-Å-long amphipathic helix 6 (residues 8-18) rests on the membrane surface 8, while the 35-Å-long trans-membrane helix 10 (residues 21-45) crosses the phospholipid bilayer 12 at an angle of 26° up to residue Lys40, where the helix tilt changes. The helix tilt accommodates the thickness of the phospholipid bilayer, which is 31 Å for E. coli membrane components.
Liposomes displaying coat protein pVIII fixed in the lipid bilayers have heretofore been prepared by sonification of the virus with excess of phospholipids, such as DMPC (dimyristoyl-sn-glycero-phosphocholine). It is also known that the pVIII protein can be reconstituted into phospholipids through a dialysis process, yielding liposomes with a lipid to protein ratio of approximately 250.
Micelle forms of the pVIII can be obtained by its complexing with different lipids, such as sodium dodecyl sulfate, dodecyl phosphatidyl choline, dihexanoyl phosphatidyl choline or lyso myristoyl phosphatidyl choline. In the micelles, like liposomes, the pVIII protein forms two a helixes connected by a hinge, amphipathic 9-mer helix (residues 8-16) accommodated in the plane of the bilayer and an 18 residue trans-membrane hydrophobic helix (residues 27-44) spans the micelle. The N and C terminal regions of the membrane protein pVIII are mobile, although the C terminus may also be involved in the helical structure. The amphipathic helix has significantly more motional freedom than the hydrophobic helix and moves on and off the micellar surface.
The instant disclosure combines the advantages of liposomes and micelles as drug delivery systems with the unique ability of landscape phages to specifically and selectively bind target sites. The inventors have developed a novel way of combining pVIII fusion phages with micelles and liposomes wherein the pVIII fusion phages display a guest peptide in every pVIII subunit.
Accordingly, a targeted drug delivery nanocarrier is provided, the nanocarrier comprising a plurality of amphipathic molecules, a targeting landscape phage and a plurality of drug molecules. The amphipathic molecules form a carrier particle having the plurality of drug molecules contained therein and the targeting landscape phage is complexed to the carrier particle. The targeting landscape phage displays a binding peptide selected to specifically and selectively bind to a target site. The desired carrier particle may be either a micelle or a liposome, or another similar, related particle. The landscape phage is preferably a filamentous landscape phage. More preferably, landscape phage is a filamentous landscape phage that displays the binding peptide in major coat protein pVIII.
The invention also contemplates a method for forming a targeted drug delivery nanocarrier. The method comprises the steps of obtaining a plurality of bacteriophage displaying a binding peptide for a desired target site, treating the plurality of bacteriophage with a denaturing agent, mixing the treated bacteriophage with a plurality of carrier particles, and purifying the mixture to obtain a targeted drug delivery nanocarrier. The denaturing agent is preferably chloroform; however, any suitable denaturing agent may be used. It is important that the denaturing agent convert the filamentous bacterial phage into a spheroid (S-form) conformation. The carrier particle is preferably a micelle or liposome, but may be any suitable carrier particle that is readily mixed with treated bacteriophage. Finally, the step of purifying preferably comprises purifying the mixture through filtration chromatography; however, any type of purifying wherein the drug delivery or nanocarriers are separated from contaminants in the mixture is acceptable according to the method of the present invention.